Local Structural Preferences and Dynamics Restrictions in theUrea-Denatured State of SUMO-1: NMR Characterization

Ashutosh Kumar,* Sudha Srivastava,* Ram Kumar Mishra,y Rohit Mittal,y and Ramakrishna V. Hosur**Department of Chemical Sciences and yDepartment of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India

ABSTRACT We have investigated by multidimensional NMR the structural and dynamic characteristics of the urea-denaturedstate of activated SUMO-1, a 97-residue protein belonging to the growing family of ubiquitin-like proteins involved in post-translational modifications. Complete backbone amide and 15N resonance assignments were obtained in the denatured state byusing HNN and HN(C)N experiments. These enabled other proton assignments from TOCSY-HSQC spectra. Secondary Ha

chemical shifts and 1H-1H NOE indicate that the protein chain in the denatured state has structural preferences in the broadb-domain for many residues. Several of these are seen to populate the (f,c) space belonging to polyproline II structure. Althoughthere is no evidence for any persistent structures, many contiguous stretches of three or more residues exhibit structuralpropensities suggesting possibilities of short-range transient structure formation. The hetero-nuclear 1H-15N NOEs are extremelyweak formost residues, except for a few at theC-terminal, and the 15N relaxation rates show sequence-wise variation. Some of theregions of slow motions coincide with those of structural preferences and these are interspersed by highly flexible residues. Theimplications of these observations for the early folding events starting from the urea-denatured state of activated SUMO-1 havebeen discussed.

INTRODUCTION

Characterization of the unfolded/denatured states of proteins

is important for many reasons; here, the term ‘‘unfolded’’

refers to a state completely devoid of any structural prefer-

ences and the term ‘‘denatured’’ refers to the lowest energy

nonnative state under a given set of conditions (1). The un-

folded and the denatured states may coincide under extreme

denaturing conditions. Firstly, the denatured state represents

the reference state for estimating the stability of a protein.

Next, the denatured state is the starting point for the folding

process in vivo. In this context, it has been a major area of

research to understand how the unfolded chain finds its final

folded state destination (2–4). There is now ample evidence

to believe that the different members of the unfolded en-

semble fold along different pathways on a so-called folding

funnel, whose broad end represents the unfolded state, and

the narrow end, the folded state (5–7). Although it is almost

impossible to know at this point of time, what the exact

nature of the denatured state in vivo is, it is believed that the

chain is not entirely random but has at least some local

preferences on the average that restrict the conformational

search by the folding chain. The local preferences could vary

depending upon the environmental conditions. In view of

this, there is vigorous effort in the literature to characterize

the denatured states created by a variety of means, which

include use of chemical denaturants (2,8–14), change of pH

conditions (15–17), or thermal denaturation (18,19). It has

been observed that different conditions populate different

conformational states in the ensemble (20,21). Among the

various means used for creating denatured states, guanidine

denaturation is known to cause the maximum degree of

unfolding. This is followed by urea and other denaturing

agents. pH denaturation appears to be more tolerant and so is

temperature. Sodium dodecyl sulphate micelles cause mostly

removal of tertiary interactions (22). Consequently, as must

be expected, the equilibrium unfolding pathways in many

of these cases have also been found to be different (23–26);

some follow a two-state process, whereas others follow mul-

tistate processes which have implications for protein stability

studies. Thus, study of all such denatured states is expected

to provide insights into the different pathways of folding,

starting from the different denatured states in the ensemble.

The unfolded state and the partially unfolded states have

also been thought to be playing important roles in protein

function. Recently, the structure-function paradigm has been

revisited and a model called the ‘‘protein-quartet model’’ has

been put forward, according to which a protein function

arises as a consequence of an interplay between folded states,

unfolded states, molten globules, and premolten globules

(27,28). A direct role of protein unfolding has been seen in

transport of proteins across the membranes (29). Lastly, the

denatured states of proteins have often been seen to cause

protein aggregation (30), and such aggregation is known to

be one of the reasons for many diseases in vivo (31,32).

One type of structure reported by NMR in denatured

proteins is a hydrophobic cluster, usually formed by local

side-chain interactions (33–36). Another type of structure

reported is a fluctuating secondary structure. In the case of

Submitted July 30, 2005, and accepted for publication December 29, 2005.

Address reprint requests to Prof. Ramakrishna V. Hosur, Dept. of Chemical

Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road,

Mumbai 400 005, India. Tel.: 91-22-2280-4545; Fax: 91-22-2280-4610;

E-mail: hosur@tifr.res.in.

Abbreviations used: HSQC, hetero-nuclear single quantum coherence;

NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect

spectroscopy; SUMO, small ubiquitin related modifier.

� 2006 by the Biophysical Society

0006-3495/06/04/2498/12 $2.00 doi: 10.1529/biophysj.105.071746

2498 Biophysical Journal Volume 90 April 2006 2498–2509

barnase (37,38), the urea-denatured protein shows regions of

fluctuating secondary structure, which correspond fairly well

to the moderately and highly structured regions of the inter-

mediates and the transition states of folding (39). Native- and

nonnative-type secondary structural preferences were seen in

the urea-denatured state of barstar (9), guanidine-denatured

state of HIV-1 protease (2,8,10), and very recently in the

acid-denatured state of hUBF HMG Box 1 (15). A nonnative

a-helical structure was observed in the guanidine denatured

state of b-lactoglobulin, an all-b-protein in the native state

(40). Similarly, b-type preferences were seen in denatured

apomyoglobin, a largely helical protein (41). In some cases,

multiple types of structures have been reported in the dena-

tured state. For example, for the drkN SH3 domain, detailed

NMR studies (42–44) indicate the presence of multiple

structures ranging from conformers with non-native structure

possessing long-range contacts to those with more compact

structures maintaining native-like secondary structure.

There is growing evidence in the literature, mostly based on

circular dichroism (CD) spectroscopy, that chemically dena-

tured proteins populate polyproline (PPII) structure which lies

in the b-domain of (f,c) space of the Ramachandran map.

This is a left-handed helical structure, generally observed in

proline-rich peptides (45–48) and has a characteristic positive

CD band at 225 nm. Further, where residual structures in the

b-domain have been detected by NMR in denatured proteins,

it is suggested that PPII structure may be populated to a sig-

nificant extent (49,50).

In view of the above, we have initiated investigation of

the structural and dynamic characteristics of the variety of

denatured states of the protein, SUMO-1, a 101-residue

protein belonging to the growing family of ubiquitin-like

proteins involved in post-translational modifications (51). It

attaches itself to many target proteins, by a process called

‘‘sumoylation’’. Before sumoylation the protein gets acti-

vated by cleaving off four residues from the C-terminal end.

There have been two reports in the literature on the structure

of the 101-residue SUMO-1 (52,53). We reported the NMR

structure of the activated SUMO-1 at pH 7.4 and 27�C (54).

All three structures were essentially similar and the average

structure of the molecule contains the following secondary

structural elements: b1: 22–28; b2: 33–39; b3: 62–66; b4:

69–72; b5: 87–93; a1: 45–55, and a2: 77–82. The b-strands

form a bent sheet. The regions intervening between second-

ary structures form flexible loops. The amino-terminal 20

residues are highly flexible. In this article, we report NMR

investigations on the characteristics of the denatured state of

SUMO-1 (1–97) created by 8 M urea at pH 5.6 and 27�C.

Hereafter, we refer to this protein as SUMO-1 only for sake

of brevity. Our results indicate that in the urea-denatured

state, a large number of residues have slight (f,c) prefer-

ences in the broad b-domain, which encompasses the ex-

tended structures as well. The dynamic motions at the high

frequency scale appear to be fairly restricted. There are

sequence-wise small variations in the milli- to microsecond

timescale motions. These results provide a description of the

denatured state on the one hand, and also provide clues to the

possible nucleation sites for the folding reaction of the pro-

tein, on the other.

MATERIALS AND METHODS

Protein preparation

SUMO-1 was prepared and purified as described elsewhere (54).

NMR spectroscopy

Isotopically labeled protein samples for NMR experiments were prepared as

described in Mishra et al. (54). The proteins were concentrated to ;1 mM

and exchanged with 0.1 M phosphate buffer (pH 5.6) containing 150 mM

NaCl, 5 mM EDTA, 1 mM DTT, and 8 M urea. The NMR experiments were

started after keeping the solutions for ;3 h so as to reach equilibrium.

All the NMR experiments were performed on a Varian Inova 600 MHz

spectrometer at 27�C (Varian, Cary, NC). A series of two-dimensional and

three-dimensional experiments (see below) were carried out which lasted for

six days. At the end of these the HSQC spectrum was again recorded to check

for the stability of the protein. We observed no change in the HSQC spectra

indicating that the protein was very stable and also had reached equilibrium

state at the beginning of the experiments. HN and 15N resonance assignments

were obtained using HNN and HN(C)N triple resonance experiments (55–

57). HNCA, HN(CO)CA, TOCSY-HSQC, and NOESY-HSQC experiments

(reviewed in (58,59)) facilitated the assignments by providing additional

checks. Standard experimental parameters were used: 24–30 ms for N-Ca and

N-C9 transfers in HNN, HN(C)N, HNCA, and HN(CO)CA; and 60–80 ms for

TOCSY transfer and 150–200 ms for NOESY transfers. The 4–16 scans were

used for each fid in the different experiments. The 40–50 and 80 complex

increments were used along the 15N and C9 dimensions respectively; 60–80

complex increments were used along the aliphatic carbon dimensions; 80–96

complex increments were used along the indirect 1H dimension. Temperature

coefficients of amide protons were measured by recording HSQC spectra at

3�C temperature increments in the range 15–36�C. 15N transverse relaxation

rates (R2) were measured using CPMG delays, 10, 50, 70*, 90, 130, 170*,

210, and 250 ms, where asterisks indicate duplicate measurement. 15N longi-

tudinal relaxation rates (R1) were measured using the inversion recovery

delays 10, 50, 120*, 220, 350, 700*, and 950 ms. Steady-state 1H-15N hetero-

nuclear NOE measurements were carried out with a proton saturation time of

3 s and a relaxation delay of 2 s. For the experiment without proton saturation

the relaxation delay was 5 s. All the relaxation experiments were carried out

using the pulse sequences described by Farrow et al. (60). HN-Ha coupling

constants were measured from a high resolution HSQC spectrum recorded

with 8192 complex t2 points and 512 complex t1 points.

Circular dichroism

Far-UV circular dichroism (CD) spectra of the protein at 27�C, were

recorded on a JASCO–J810 spectropolarimeter (Jasco, Hachioji, Japan), at 0

and 8 M urea concentrations using 0.2 cm cell. The protein concentration

was 20.7 mM. The samples at the appropriate conditions were equilibrated

for at least 10–12 h before CD measurements. Each spectrum was an average

of eight scans (slit width of 2 nm).

RESULTS AND DISCUSSION

Resonance assignments

Resonance assignment in denatured proteins by the standard

triple resonance experiments such as HNCA, HN(CO)CA,

NMR Characterization of Denatured SUMO-1 2499

Biophysical Journal 90(7) 2498–2509

HNCACB, CBCANH, CBCA(CO)NH, etc., which have

proved extremely successful for folded proteins, is severely

hampered because of the high degeneracy of the amide and

carbon chemical shifts (61). Consequently there have been

relatively few proteins on which detailed studies at residue

level have been carried out (61–65). In such situations, recent

experiments that exploit the 15N or CO chemical shifts,

which have good dispersions in both folded and unfolded

proteins, would be extremely valuable (65,66). Presently, we

were able to obtain complete amide proton and backbone15N assignments for SUMO-1, at pH 5.6 and 27�C, in 8 M

urea using the HNN and HN(C)N experiments described

by us a few years ago (55–57). These exploit the good

dispersion of the 15N chemical shifts along two of the three

dimensions, and also provide many checkpoints along the

sequential walk by way of special patterns of peaks around

Gly and Pro residues. Thus side-chain identification does not

become very essential during the sequential walk, and this

enhances the speed of assignment. However, under rare occa-

sions of overlaps, TOCSY-HSQC spectrum, which provides

the spin system information, helps resolve the ambiguities.

An illustrative sequential walk through the HNN spectrum of

SUMO-1 at 27�C, pH 5.6 is shown in Fig. 1 A and the

summary of all the sequential connectivities is shown in Fig.

1 B. Fig. 2 shows the 1H-15N HSQC spectrum with all the

peak assignments. During the course of these sequential as-

signments, many of the side-chain proton assignments were

obtained from the TOCSY-HSQC spectrum.

Structural propensities in the denatured state

Deviations of observed chemical shifts from the random coil

values (secondary shifts) are very good indicators of sec-

ondary structures in proteins. The random coil values are

generally measured using short 5–6 residue peptides, as they

do not possess any structural preferences in solution. These

are particularly useful in denatured proteins where NOEs are

rather scarce. However, there have been more than one set of

random coil values reported in the literature and these differ

due to slightly different experimental conditions. We have

calculated these shifts using two sets, one by Wishart and

Sykes (67,68) and the other by Schwarzinger et al. (69). The

former uses pH 5 and 1 M urea, while the latter uses 8 M urea

and pH 2.3 for the measurements on peptides, GGXGG,

where X is the residue of interest for random coil chemical

shift and G is glycine. In our case the two calculations made

only a marginal difference and we decided to use the data

given by Schwarzinger et al. (70), since these authors have

also described a set of sequence corrections to the random

coil values for all the nuclei.

Among the various secondary chemical shifts (Dd), those

of Ha, 13Ca, and 13CO are the most diagnostic of structural

information in the polypeptide chain (63,64). Thus if some

residues show positive (downfield) Ha and negative (upfield)I3Ca and I3CO structure-dependent chemical shift deviations

(secondary shifts, Dd), then those residues are taken to have

(f,c) preferences in the b-domain of the Ramachandran map

FIGURE 1 (A) Sequential walk through the F1–F3

planes of HNN spectrum of SUMO-1 in 8 M urea at pH

5.6 and 27�C. Sequential connectivities are shown for

Gly-19 to Leu-24 stretch. The F2 (15N) values are

shown at the top for each strip. The black and red con-

tours indicate positive and negative peaks. The distinct

Gly plane serves as the start point in the sequential

assignment. (B) Summary of sequential assignment

obtained from HNN walk along the primary sequence

of SUMO-1. Gly shown in green worked as start/check

point; Pro shown in red worked as break point.

2500 Kumar et al.

Biophysical Journal 90(7) 2498–2509

((f,c) ¼ (�70 to �150�, 90–180�)). Residues with a-helical

propensities ((f,c) ¼ (�40 to �80�, �30 to �60�)) show

the opposite pattern.

We have used Ha secondary shifts to characterize the

residual structure in the protein. Fig. 3 A shows a plot of the

secondary shifts against the sequence for SUMO-1 at pH 5.6

and 27�C. Clearly, from the pattern of the deviations which

show a positive bias, the chain does not appear to be a

random coil. Though the shifts are small, overall there seems

to be some tendency for a large number of residues across the

chain to populate the broad b-region of the (f,c) space; this

includes the extended conformations and PPII structures

((f,c) ¼ (�70 to �80�, 140 to 150�)) as well. There are a

few contiguous stretches of three or more residues showing

positive deviations of .0.08 ppm and these may be taken to

indicate formation of b-structures, perhaps transiently, along

the chain. They have been indicated by solid horizontal bars

inside the figure.

Next, we measured 3J (HN- Ha) coupling constants for all

the residues. Three bond HN-Ha coupling constants are

useful indicators of f-torsion angles. This coupling constant

is of the order of 3–5 Hz for a helical structure (including

PPII) and 8–11 Hz for a b-structure (64). For a random coil, a

weighted average of these values would be observed, which

is typically between 6.0 and 8.0 Hz for most residues. It is

also observed that the random coil value for any residue is

influenced by its N-terminal neighbor and thus two sets of

values have been reported for each residue, depending upon

whether the N-terminal neighbor belongs to one of the two

groups of residues (group I: F, W, H, Y, I, T, V, and group II:

remaining residues except glycine) (71). Thus, under any

given experimental conditions, one can calculate deviation

of the observed coupling constant from the sequence-

dependent random coil value, (Jobs–Jrc), for every residue.

These ‘‘secondary coupling constants,’’ as we call them,

throw valuable light on the secondary structural propensities

along the polypeptide chain. Negative secondary coupling

constants would indicate helical propensities (including PPII)

and positive secondary coupling constants would indicate

b-propensities (71). This led us to the idea that a combined

use of secondary chemical shifts and secondary coupling con-

stants would enable distinction between b and PPII struc-

tures. For a truly b-structure, both the secondary shifts and

the secondary coupling constants would be positive, whereas

for the PPII helix the secondary coupling constant would be

negative, as in the common right-handed a-helices, and the

secondary shifts would be positive, like in the b-structures.

The measured values of the secondary coupling constants

from a high resolution HSQC spectrum of the protein (an

illustrative region to show the quality of the spectral reso-

lution is shown in Fig. 3 B), are shown in Fig. 3 C. The

deviations are mostly ,1.0 Hz for most residues. We may

mention here that the accuracy of secondary coupling con-

stant estimation is ;0.5 Hz for the positive values, and

FIGURE 2 (1H-15N) HSQC spec-

trum of SUMO-1 in 8 M urea at pH

5.6 and 27�C. Residue-specific assign-

ment for each peak is marked on the

spectrum.

NMR Characterization of Denatured SUMO-1 2501

Biophysical Journal 90(7) 2498–2509

slightly worse but ,;1 Hz for negative deviations; this is

because the negative deviations occur when the coupling

constants themselves are small. Thus the deviations beyond

these values may indicate slight (f,c) preferences. Interest-

ingly, most of the residues exhibiting negative secondary

coupling constants have positive secondary shifts, suggest-

ing that their (f,c) values may populate the PPII structure to

a reasonable extent. A residue-wise assessment of the b or

extended, and PPII preferences, from a combined use of sec-

ondary shifts and secondary coupling constants, is given in

Table 1 of the Supplementary Material. The fact that the

residues exhibiting PPII type propensities are not conti-

guous along the chain indicates that there is no stable PPII

helix formed in the ensemble. To check on this further, we

recorded the CD spectrum at 27�C in 8 M urea and this is

shown along with a similar spectrum in the absence of urea

to serve as a reference, in Fig. 3 D. Clearly, there is no char-

acteristic positive band at 225 nm confirming the absence of

a stable PPII helix in the denatured state ensemble.

The amide proton temperature coefficients (negative val-

ues with magnitudes ,;4.5 ppb/K) indicate hydrogen bond-

ing and thus report on the presence of persistent structures

FIGURE 3 (A) Sequence-corrected secondary chemical shift for Ha (Dd(Ha)) in SUMO-1 at 8 M urea at pH 5.6 and 27�C. The sequence-corrected random

coil values used are those determined by Schwarzinger et al. (70). The native secondary structural elements are shown on the top of the figure and the segments

showing structural propensities are indicated by solid bars. (B) Selected region from a high resolution (1H-15N) HSQC spectrum of SUMO-1 in 8 M urea at pH

5.6 and 27�C. The splitting in the peaks was used to measure 3J (HN-Ha) coupling constants. (C) Secondary coupling constants (see text) are plotted against

sequence for SUMO-1. (D) Far-UV CD spectra of SUMO-1 at pH 5.6 and 27�C, in the absence of urea (shaded circles), and in 8 M urea (open circles).

2502 Kumar et al.

Biophysical Journal 90(7) 2498–2509

(72). For random coils the temperature coefficients are

roughly in the range �6 to �9.5 ppb/K for the different

residues (73). We measured the residue-wise amide proton

temperature coefficients of the protein from HSQC spectra

recorded in the temperature range 15–36�C. Since all the

measured values were negative we calculated their devia-

tions from the random coil values by taking magnitudes

only, and these results are shown in Fig. 4. We see that the

observed deviations D(DdNH/DT) are largely positive. From

the absence of significant negative deviations, it is clear that

there are no stable intramolecular H-bonds formed in most

regions of the polypeptide chain. This rules out the possi-

bility of persistent well-defined structural elements like

a-helix, b-sheet, b-turn, etc., in the denatured state.1H-1H NOEs in folded proteins exhibit secondary struc-

ture specific patterns. b-structures including PPII and type II

turns are characterized by strong Ha(i) – HN(i11) NOEs,

whereas a-helices are characterized by strong HN(i) –

HN(i11) NOEs (74). The a-helices also produce medium

range NOEs, from HN (i) to Ha (i�3) and Ha(i�4). In ad-

dition, several other NOEs will also be seen from the amide

protons to other intraresidue protons. In unfolded proteins,

however, the NOEs are generally weak in the first place due

to greater flexibility of the chain, and the above selectivity

with regard to secondary structure may be lost due to mea-

surably good population of the different secondary structure

types in the ensemble. However, if one does find any kind

of preference in the NOE patterns, then that can be taken to

indicate a higher population of those specific structures. In

SUMO-1 denatured by 8 M urea, the 1H-1H NOEs in the

NOESY-HSQC spectra recorded with a mixing time of

150 ms, are rather sparse, but, interestingly, do show some

pattern. These are shown in Fig. 5, which seem to support the

conclusions derived above from the secondary shifts. A ma-

jor portion of the chain shows fairly intense sequential Ha(i)– HN(i11) NOEs, and only a few of them also show self-

NOEs. In a dipeptide, the sequential distance, daN(i, i11) is

sensitive to torsion angle ci and varies from 2.2 to 3.5 A�, the

shortest distance occurring for ci in the b-domain. Likewise,

the distance between Ha and HN within the same residue,

daN(i, i), which is sensitive to the torsion angle fi, varies

within a narrow range from 2.2 to 2.9 A� (74). Thus, for

those residues where only the sequential peaks are seen, the

(f,c) propensity has a preponderance of theb-structure, which

has the shortest sequential distance and the longest intra-

residue distance. For those residues where both the peaks are

observed, the fi may show slight preference toward a shorter

intraresidue distance, still within the b-domain. From the NOE

data, we conclude that the regions, 9–15, 17–33, 43–48, 61–

68, 71–74, 82–87, and 90–95 have a higher population of

b-structures in the ensemble.

All the above observations taken together indicate that the

SUMO-1 polypeptide chain in 8 M urea does not possess any

stable secondary structures, but has some propensity for for-

mation of transient structures with (f,c) preferences in the

broad b-domain, in 4–5 regions. Several residues in these

regions seem to populate the (f,c) space belonging to the

PPII structure. The structural propensities presently observed

in the 8 M urea-denatured state encompass both native and

nonnative type preferences for the individual residues, and

this would have implications for the folding mechanisms of

the protein.

Dynamics restrictions

Since the protein chain exhibited some local structural pref-

erences in the denatured ensemble, it is intuitively natural to

expect some variations in the motional characteristics along

the length of the chain as well. To probe this explicitly, we

carried out 15N R1, R2 and heteronuclear 1H-15N NOE

measurements, which throw light on the motions over a wide

range of timescales (75–77). Among these, the NOEs are

very sensitive to picosecond timescale motions, R2 values are

sensitive to milli- to microsecond timescale motions (con-

formational transitions) and the R1 values are sensitive to both

low and high frequency motions (nanosecond-to-picosecond

timescale motions). The heteronuclear NOE intensity varies

from ;�4 to 0.9 depending upon the motional correlation

times (75,78). Negative values indicate high frequency

(picosecond timescale) large amplitude motions, whereas

positive values indicate nanosecond timescale motions. In

the denatured states, where there is heterogeneity of the con-

formational states and of the correlation times, these relax-

ation and NOE data can only be interpreted qualitatively to

derive relative motional trends along the chain. Reduced

spectral density analysis based on 15N R1, R2 and heteronu-

clear 1H-15N NOE is the best suited method for studying

motions at residue level in denatured proteins (15,64). How-

ever, in the present case this was hampered by the absence of

measurable 1H-15N NOEs due to their low intensities (see

below); this of course must have a bearing on the motional

characteristics themselves. Nevertheless, we have been able

FIGURE 4 Secondary temperature coefficients. Deviation of amide pro-

ton temperature coefficients from the random coil temperature coefficient

(magnitude mode) at pH 5.6 plotted against the sequence. The native

secondary structure elements are indicated at the top.

NMR Characterization of Denatured SUMO-1 2503

Biophysical Journal 90(7) 2498–2509

to obtain some useful insights into these motions, especially

for the nanosecond timescale components and the confor-

mational transitions, from a qualitative analysis of the R1, R2

relaxation rates.

Fig. 6 shows 1H-15N correlation spectra recorded during

the 1H-15N steady-state NOE measurements. The spectrum

in Fig. 6 A is obtained without irradiation of the protons

and the spectrum in Fig. 6 B is the one recorded with pro-

ton irradiation of 3 s to reach the steady state. Although the

normal spectrum is similar to the typical HSQC spectrum,

the spectrum in Fig. 6 B shows only four peaks; the peaks

have also good intensities, as can be seen from the cross sec-

tion for the weakest of the peaks. This is somewhat unusual

compared to similar studies on other denatured proteins

where the HSQC spectra recorded in the presence of proton

saturation have good peak intensities and a distribution of

positive and negative NOEs has been seen. As a reference,

we show in Fig. 6 C the spectrum on folded SUMO-1 (pH

7.4, 27�C), which was recorded with proton irradiation and

the same protein concentration as in A and B. This spectrum

has clearly good intensities. Thus the present observation of

only four discernible peaks in spectrum B in Fig. 6 must have

dynamic implications. Firstly, two of the four peaks have

greater intensity than the corresponding ones in spectrum A,

and this can happen only if the 1H-15N NOEs are negative.

All the peaks in spectrum B have the same phase and there-

fore the other two peaks also must be negative in sign. As

marked, these peaks belong to the C-terminal of the protein,

and the negative sign indicates that this segment is highly

mobile, exhibiting high amplitude picosecond timescale

motions. This, however, is not surprising, since the terminal

residues generally have been seen to exhibit a greater

flexibility than the other residues. More importantly, the fact

that no other peak is seen in spectrum B, suggests that the

NOEs are very small, perhaps below the noise level and a

simple explanation for this could be that the rest of the entire

FIGURE 5 Summary of sequence-specific1H-1H connectivities obtained from the NOESY-15N-1H HSQC spectrum.

FIGURE 6 Spectra showing 15N-1H heteronu-

clear NOE of SUMO-1 in 8 M urea at pH 5.6 and

27�C. (A) Reference spectrum without irradia-

tion of protons; (B) spectrum with irradiation of

protons for 3 s; and (C) spectrum of the folded

protein (pH 7.4, 27�C) with proton irradiation

as in B. In B, a cross section through the peak

for Q94 (Q94 is enclosed in a box) is shown to

demonstrate the good sensitivity in the spectrum.

The red and black contours indicate negative and

positive peaks.

2504 Kumar et al.

Biophysical Journal 90(7) 2498–2509

chain is relatively more restricted, i.e., the amplitudes of the

high frequency motions are small. And, the correlation times

for the individual N-H vectors (in the nanosecond regime)

may also be such that the steady-state NOEs are nearly zero.

Fig. 7 A shows residue-wise R1 values in the SUMO-

1 protein at 27�C. The average relaxation rate is ;1.69 s�1,

which is typically the value one sees for this size protein.

Interestingly, however, we see some sequence-dependent

variations along the chain, some of which seem to coincide

with the expected motional behaviors in the native state.

For example, the segments/residues (residues 29–31, 40, 60)

belonging to the loops between the native secondary struc-

tures (b1: 22–28; b2: 33–39; b3: 62–66; b4: 69–72; b5: 87–

93; a1: 45–55; and a2: 77–82), which should be more

flexible than other regions, have indeed significantly larger

than average R1 values, indicating larger nanosecond-

timescale motions; these residues are identified by solid

circles in the figure. However, this is not true for other

flexible regions of the native protein, especially at the long

N-terminal segment. These seem to suggest that just as one

can have native- and nonnative-type structural preferences,

there can also be native and nonnative type motional char-

acteristics in the denatured state.

The R2 data shown in Fig. 7 B also displays interesting

sequence-wise variations. In the denatured states, almost

every residue would have some contribution from conforma-

tional exchange (Rex) to its transverse relaxation rate,

R2 ¼ R2;int 1Rex;

where R2,int is the intrinsic transverse relaxation rate. How-

ever, there can be subtle variations in these due to possi-

bilities of differences in short-range interactions because of

sequence effects and consequent different local structural pref-

erences. Thus deviations from the average values, though small

as they would be, can be interpreted to suggest different de-

grees of conformational exchange contributions to the trans-

verse relaxation rates. In Fig. 7 B, among the many residues/

short stretches that have higher than average R2 (3.66 s�1)

values, we see that the stretches, 19–26 (average R2 is 4.17

s�1), and 89–92 (average R2 is 4.12 s�1), are at least four

residues long and coincide with the regions of structural

propensity derived from the secondary chemical shifts (this

data from Fig. 3 A is included in the figure here for ready

comparison). Hence, these regions may be considered to

execute slow segmental motions. The segment 19–26 over-

laps with the native b1 strand and the segment 89–92 over-

laps with the native b5 strand. Furthermore, the loop 29–31

between the b1 and b2 strands that exhibited high R1 value

also shows high R2 values. This indicates, firstly, that the

slow motions representing conformational exchange and the

nanosecond-scale motions of the N-H vectors are indepen-

dent. Secondly, since the b1 and b2 strands interact in the

native structure to form the antiparallel b-sheet, we are

tempted to think that the slow motions in this region help to

facilitate such an interaction in the denatured state as well.

However, as both the strands have some residues with PPII

propensities, the transient structure formation may not be

entirely native-like.

Among the residues that have lower than average R2

values signifying greater flexibilities, special mention must

be made of K37 and S61, which are in the interior of the

chain and have conspicuously low R2 (;2 s�1) values. Such

observations have been made in the past in at least two cases,

namely, apomyoglobin (36) and HIV-1 protease (2) and they

have been taken to indicate molecular hinges along the poly-

peptide chain. Interestingly, in SUMO-1 these residues are

seen to be near the edges of those areas that have exhibited

higher b-propensities along the chain, consolidating their

role as molecular hinges. Another interesting feature notice-

able in the R2 data in Fig. 7 B is that the N-terminal and the

C-terminal residues have distinctly different motional be-

haviors, the C-terminal residues having relatively greater

flexibility.

Implications to protein folding

As pointed out by several authors, the characteristics of the

denatured state have a significant influence in directing the

folding process of a protein (79–83). A denatured state under

some conditions may correspond to an intermediate along

the refolding pathway of a protein (1). The residual structural

propensities in the denatured state are suggestive of initial

FIGURE 7 15N relaxation rates plotted against sequence for SUMO-1 in

8 M urea at pH 5.6 and 27�C. (A) Longitudinal (R1) and (B) transverse (R2)

relaxation rates. The structural propensities seen in Fig. 3 A are also indi-

cated in B with solid bars for ready reference. Solid circles in A and B

identify residues with high flexibility.

NMR Characterization of Denatured SUMO-1 2505

Biophysical Journal 90(7) 2498–2509

folding events along the chain and its dynamic characteris-

tics throw further light on these events. There may be local

and nonlocal interactions and therefore, native and nonnative

propensities as well. Although the local interactions essen-

tially define intrinsic preferences, the nonlocal interactions

would define the topologies. The existence and significance

of nonlocal preferences under strong denaturing conditions,

however, remains a controversy (41,50). For those proteins,

where there is an initial collapse of the polypeptide chain

when refolding is initiated by dilution of denaturant, the

collapse is likely to be a consequence of transient interac-

tions between hydrophobic clusters that persist in the dena-

tured state (33,34,36). In the case of barnase, which exhibits

a fluctuating secondary structure in the denatured state, it is

suggested that folding is initiated around the native-like local

structures (38). In b-lactoglobulin, it was observed that the

early folding intermediate contained a nonnative helix and

this was attributed to the intrinsic high helical preference

of the amino acid residues involved in the denatured state

(40,84).

In SUMO-1 we observe that the protein chain in the urea-

denatured state has (f,c) preferences in the b-domain of the

Ramachandran map in many segments. Several of these are

native-type preferences. Many residues exhibit (f,c) pref-

erences corresponding to the PPII helix. However, there is no

indication of stable secondary structure in the denatured

ensemble. From the dynamics data it appears that in the urea-

denatured state the protein chain is not randomly fluctuating,

but exhibits sequence dependent motional variations. Inter-

estingly, the segment (residues 29–31) exhibiting larger nano-

second timescale motions coincides with the flexible loop

between b1 and b2 strands in the native structure. Although

this is not true for all the flexible regions of the native

structure, it seems to suggest the existence of some native-

type motional signature in the denatured state. The structural

and dynamics results provide extremely valuable clues to the

initial folding events in the protein. Although the structural

preferences indicate possible nucleation sites for the folding

process, the relaxation data indicate how motions can facil-

itate interactions between segments in directing the folding

process. Specifically, the slow conformational exchange ex-

hibited by the loop between b1 and b2 strands may promote

an interaction between the two strands; these are involved in

the formation of an antiparallel b-sheet in the native state.

However, few residues in each of these strands have PPII

propensities and thus the resultant interaction may not be

entirely native-like in the denatured state. Further, residues

K37 and S61, which exhibit greater flexibilities, are seen to

flank regions of higher structural propensities. Thus, they

may serve as molecular hinges for motions of the neighbor-

ing segments that may be transiently ordered in the en-

semble. All these lead to an implication that, possibly, the

conformational transitions and the hinged motions facilitate

short range structure formations, though transiently, in the

denatured state, and thus may serve as early folding events.

Parallel folding events from other regions such as b3, b5,

which also exhibit structural propensities and slow confor-

mational transitions, are also conceivable.

SUMO-1 belongs to the ubiquitin family of proteins, and

shares the same fold topology and the secondary structural

architecture, bbabbab. Thus, it would be interesting to

compare the above results with the folding studies on frag-

ments of ubiquitin reported in the recent past (85–87). It was

observed that the peptide, 1–17, representing the b1-loop-b2

hairpin in ubiquitin, formed a stable nativelike b-hairpin,

which suggested that the peptide segment has an intrinsic

tendency to form such a structure (86). On this basis, the

authors proposed that b1-loop-b2 hairpin formation may

be an initial folding event in the folding mechanism of

ubiquitin. Interestingly, the structural and dynamic prefer-

ences in the denatured state of full-length SUMO-1 protein

described in this work also indicate the above segment as the

folding initiation site. On the other hand, the experiments

with the C-terminal peptide (residues 36–76) of ubiquitin

exhibited nonnative a-helical preferences (87). In SUMO-1

we found some nonnative structural preferences in the broad

b-domain in the N-terminal (residues 2–6, 17–21), and in the

center (residues 48–53) along the protein chain. Thus it

appears that the initial folding events in both the proteins

have a combination of native- and nonnative-like events, but

the finer details have some events in common as well as

some differences. This may not be too surprising considering

that the two proteins have only 18% sequence homology and

consequently the interactions directing the folding process

could have differences.

CONCLUSIONS

In conclusion, we have successfully investigated here, using

a variety of NMR probes, the nature of the urea-denatured

state of SUMO-1, an important protein involved in post-

translational modifications. This became possible because

of the availability of new experimental protocols recently de-

veloped by us for obtaining resonance assignments in un-

folded proteins where the chemical shift dispersion of amide

protons and carbons is poor. Our data indicates that the pro-

tein in the urea-denatured state exhibits structural prefer-

ences in the broad b-domain of the Ramachandran map, over

the major length of the chain. Several residues populate the

(f,c) space corresponding to the PPII helix. The polypeptide

chain has significant motional restrictions and these show

sequence-dependent variations. As the structural propensi-

ties and the motional characteristics in the denatured state

have a major influence on the folding pathways of proteins,

the present observations suggest that, in SUMO-1, folding

from the urea-denatured state may get initiated around the

b1-loop–b2-region along the polypeptide chain. This region

has not only a contiguous stretch of b-propensities, but also

contains many residues exhibiting significant conformational

transitions. A few residues at the edges of these segments

2506 Kumar et al.

Biophysical Journal 90(7) 2498–2509

with structural propensities exhibit high flexibility and thus

may contribute to what one may call hinge motions facili-

tating segment movements and transient structure forma-

tions. Early folding events in a few other regions such as

b3, b5 strands are also conceivable, which would represent

parallel folding processes.

SUPPLEMENTARY MATERIAL

An online supplement to this article can be found by visiting

BJ Online at http://www.biophysj.org.

We thank the Government of India for providing financial support to the

National Facility for High Field NMR at the Tata Institute of Fundamental

Research.

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NMR Characterization of Denatured SUMO-1 2509

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